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First published online September 13, 2007
Stem Cells Vol. 25 No. 12 December 2007, pp. 3194 -3203
doi:10.1634/stemcells.2007-0402; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Fas Transduces Dual Apoptotic and Trophic Signals in Hematopoietic Progenitors

Michal Pearl-Yafea, Jerry Steina, Esma S. Yolcub, Daniel L. Farkasc, Haval Shirwanb, Isaac Yaniva, Nadir Askenasya

aFrankel Laboratory, Center for Stem Cell Research, Department of Pediatric Hematology-Oncology, Schneider Children's Medical Center of Israel, Petach Tikva, Israel;
bInstitute for Cellular Therapeutics and Department of Microbiology and Immunology, University of Louisville, Louisville, Kentucky, USA;
cMinimally-Invasive Surgical Technologies Institute, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA

Key Words. Apoptosis • Bone marrow transplantation • Differentiation • Fas

Correspondence: Nadir Askenasy, M.D., Ph.D., Frankel Laboratory, Center for Stem Cell Research, Schneider Children's Medical Center of Israel, 14 Kaplan Street, Petach Tikva, Israel 49202. Telephone: 972-3921-3954; Fax: 972-3921-4156; e-mail: anadir{at}012.net.il

Received on June 15, 2007; accepted for publication on August 29, 2007.

First published online in STEM CELLS EXPRESS  September 13, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Stem cells and progenitors are often required to realize their differentiation potential in hostile microenvironments. The Fas/Fas ligand (FasL) interaction is a major effector pathway of apoptosis, which negatively regulates the expansion of differentiated hematopoietic cells. The involvement of this molecular interaction in the function of hematopoietic stem and progenitor cells is not well understood. In the murine syngeneic transplant setting, both Fas and FasL are acutely upregulated in bone marrow-homed donor cells; however, the Fas+ cells are largely insensitive to FasL-induced apoptosis. In heterogeneous populations of lineage-negative (lin–) bone marrow cells and progenitors isolated by counterflow centrifugal elutriation, trimerization of the Fas receptor enhanced the clonogenic activity. Inhibition of caspases 3 and 8 did not affect the trophic signals mediated by Fas, yet it efficiently blocked the apoptotic pathways. Fas-mediated tropism appears to be of physiological significance, as pre-exposure of donor cells to FasL improved the radioprotective qualities of hematopoietic progenitors, resulting in superior survival of myeloablated hosts. Under these conditions, the activity of long-term reconstituting cells was not affected, as determined in sequential secondary and tertiary transplants. Dual caspase-independent tropic and caspase-dependent apoptotic signaling place the Fas receptor at an important junction of activation and death. This regulatory mechanism of hematopoietic homeostasis activates progenitors to promote the recovery from aplasia and converts into a negative regulator in distal stages of cell differentiation.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Stem and progenitor cells are often required to perform differentiation tasks under extreme conditions of injury and inflammation. In the bone marrow transplant setting, aggressive chemotherapy and radiation inflict severe injury to the host bone marrow. In the aftermath of ablative injury, donor hematopoietic stem and progenitor cells (HSPC) find their way to the host bone marrow, where they seed and engraft to reconstitute the immune-hematopoietic system. In this process, the expression and activation of death receptors in the developing hematopoietic cells have been assigned various functional roles, in particular negative regulation of differentiated cells; however, the involvement of the death receptors in the proximal stages of HSPC function is unclear. The mechanisms by which hematopoietic reconstituting cells flourish in such a devastated environment is of particular interest, as they may be used to improve the efficiency of engraftment.

An early study reported an important role of tumor necrosis factor-{alpha} (TNF-{alpha}) in suppression of donor cell activity after human bone marrow transplantation [1]. Subsequently, the role of apoptotic signals in regulation of the hematopoietic system has attracted much attention [2–4]. In vitro assays have assigned the hematopoietic growth factors an important role in inhibition of apoptosis [5]. Under various experimental conditions, TNF-{alpha} has dual activity as a promoter and suppressor of hematopoietic progenitor function [6–8]. Subsequent studies have shown that TNF-{alpha} synergizes with interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in support of proliferation and suppresses the effect of granulocyte colony-stimulating factor and stem cell factor (SCF) [7–10]. In addition to the direct proapoptotic effect of TNF through its two receptors, this cytokine was shown to induce the expression of Fas in human and murine HSPC [11–18] as a feature of cross-talk between the TNF superfamily receptors [19]. The expression of death receptors was assigned a negative regulatory role in maintenance of the pool of hematopoietic progenitors under physiological conditions and in the transplant setting [20–22]. This contention is particularly relevant to Fas, which appears to be a ubiquitous response of HSPC activation that is coupled to cell cycling and is induced by a variety of growth factors and injury cytokines [13–17, 20–22]. We therefore focused on the function of the Fas receptor as a putative common executioner of apoptosis in murine hematopoietic progenitor cells.

Fas-mediated apoptosis is an infrequent event in hematopoietic progenitors under steady-state operational conditions. The first reason is the low level of Fas expression in murine and human HSPC derived from fetal and adult tissues [22–25]. The second reason is the elevated level of antiapoptotic factors, including FLICE inhibitory protein (FLIP), Bcl-2, and survivin, which protect primitive HSPC from apoptosis [26–29]. Thus, hematopoietic progenitors are insensitive to death signals because of the absence of the appropriate receptors, and even if they sense the death signals, they do not easily succumb to apoptosis. The Fas receptor is expressed in proliferating and differentiating hematopoietic progenitors, playing an important role of negative regulation of distal differentiation in all hematopoietic lineages [30–36].

Several in vivo and in vitro studies have demonstrated variable sensitivities of hematopoietic precursors to Fas-mediated apoptosis, including impaired viability and reduced clonogenesis [10–17]. In murine HSPC, TNF-induced Fas expression reduced the engraftment efficiency and negatively regulated the self-renewal of highly purified progenitors [37]. Consistently, the clonogenic activity of progenitors was significantly reduced by exposure to an activating anti-Fas antibody (Jo2), an effect that was markedly enhanced by TNF-{alpha} and was partially reversed by transforming growth factor-β1 [22, 37–39]. Although the TNF-induced deficits in all these hematopoietic cell functions have been attributed both to direct proapoptotic effects and indirectly to induced Fas expression, some experimental evidence questions the validity of the indirect (Fas-mediated) mechanism. In murine HSPC, the cells with best hematopoietic reconstituting potential are Fas-positive [37], and ~50% of the colony-forming cells in spleen-derived lineage-negative (lin–) progenitors were resistant to Fas ligation [40]. In addition, the effect of TNF-{alpha} was related to cell cycling, in a Fas-independent manner, since HSPC from wild-type and lpr mice were equally sensitive to TNF-mediated suppression [37]. Additional recent experimental evidence challenges the putative negative role attributed to Fas in the murine transplant setting. Hematopoietic progenitors that homed successfully to the bone marrow showed a remarkable upregulation of the Fas receptor and Fas ligand (FasL), accompanied by absolute resistance to an apoptotic challenge consisting of membranous FasL protein [25]. Furthermore, ectopic expression of FasL was efficient in abrogating alloimmune responses and improved the efficiency of lin– bone marrow cells in syngeneic transplants [41]. Taken together, these data suggest that the subset of engrafting progenitors are insensitive to Fas-mediated apoptosis and question whether the receptor has trophic attributes in the transplant setting. Therefore, we assessed trophic functions mediated by Fas and used caspase inhibition to distinguish between death and growth signals in naïve murine progenitors. We found that Fas transduces growth signals in hematopoietic progenitors, a process that requires receptor trimerization and does not involve caspase activation.


   METHODS
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Animal Preparation and Transplantation
Mice used in this study were C57Bl/6J (B6, H2Kb, CD45.2), B6.SJL-Ptprca Pepcb/BoyJ (H2Kb, CD45.1), B6.MRL-Faslpr/J (lpr, H2Kb, CD45.2), C57BL/6-TgN(ACTbEGFP)1Osb (GFP, H2kb, CD45.2), and BALB/c (H2Kd), purchased from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). The mice were housed in a barrier facility. All procedures were approved by the Institutional Animal Care Committee. Recipients were conditioned by sublethal (850 rad) and lethal (950 rad) total-body irradiation using an x-ray irradiator (RadSource 2000; Rad Source Technologies, Inc., Alpharetta, GA, http://www.radsource.com) at a rate of 106 rad/minute. Irradiation was routinely performed 18–24 hours before transplantation. Notably, x-ray irradiation is different from {gamma}-irradiation in myeloablative dose and toxicity. For transplantation, cells suspended in 0.2 ml of phosphate-buffered saline (PBS) were infused into the lateral tail vein.

Cell Isolation, Characterization, and Staining
Whole bone marrow cells (wBMC) were harvested, and lin– cells were isolated by immunomagnetic separation as previously reported [25, 41]. The efficiency of the lin– cell separation procedure was reassessed by flow cytometry using a cocktail of fluorescein isothiocyanate-labeled monoclonal antibody (mAb) against the lineage markers (eBioscience Inc. [San Diego, http://www.ebioscience.com] and BD Pharmingen [San Diego, http://www.bdbiosciences.com/index_us.shtml]). Subsets enriched in stem cells with long-term hematopoietic reconstituting potential (LTR) and progenitors with short-term reconstituting potential (STR) were isolated by counterflow centrifugal elutriation using the J-6 rotor of a Beckman centrifuge (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com) [42–45]. wBMC harvested from femurs and tibia were fractionated at flow rates of 15, 25 (Fr25), 29, and 33 ml/minute at 3,000 rpm and in the rotor-off (R/O) position (STR). Fr25 cells were lineage-depleted by incubation at 4°C with rat anti-mouse mAb against AA-4, CD5, GR-1, Mac-1, and B220 extracted from hybridoma cell lines (American Type Culture Collection, Manassas, VA, http://www.atcc.org) and purified TER119 (eBioscience) to obtain the Fr25 lin– fraction (enriched in LTR cells). The efficiency of lineage depletion of the Fr25 lin– cells was reassessed by flow cytometry using a cocktail of fluorochrome-labeled mAb.

Flow Cytometry
Measurements were performed with a Vantage SE flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Before analysis, the peripheral blood and bone marrow cells were layered over 1.5 ml of lymphocyte separation medium at room temperature (Cedarlane Laboratories, Ltd., Burlington, ON, Canada, http://www.cedarlanelabs.com). After centrifugation, the low-density cells were aspirated from the interface, washed in PBS, and incubated for 45 minutes at 4°C with labeled primary mAb or counterstained with a fluorochrome-labeled secondary mAb. Donor chimerism in syngeneic transplants was determined from the percentage of donor and host peripheral blood lymphocytes using monoclonal antibodies against minor antigens CD45.1 (clone A20; eBioscience) and CD45.2 (clone 104; eBioscience), respectively. Cell death and apoptosis was determined in cells incubated with 5 µg/ml 7-aminoactinomycin D (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and annexin-V (IQ Products, Groningen, The Netherlands, http://www.iqproducts.nl). The Fas receptor was identified with a primary labeled antibody (clone 15A7; eBioscience), and FasL was stained with a biotinylated MFL4 mAb (BD Pharmingen) and fluorochrome-labeled streptavidin (eBioscience). Positive staining was determined on a log scale, normalized with control cells stained with isotype control antibodies.

Apoptotic Challenge Using FasL Protein
A20 murine lymphoblastoma cells and splenocytes served as controls for the apoptotic activity of a chimeric protein composed of core streptavidin and metalloproteinase cleavage site-deficient FasL [25, 46, 47]. Bone marrow cells were incubated (5 x 106 cells per milliliter) for 24 hours in {alpha}-MEM culture medium (Biological Industries, Beit Haemek, Israel, http://www.bioind.com) supplemented with StemPro Nutrient Supplement (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), 2 mM L-glutamine, 50 µM 2β-mercaptoethanol (2β-ME), 10 ng/ml SCF, and 100 ng/ml thrombopoietin (TPO). All chemokines were purchased from Peprotech (Rocky Hill, NJ, http://www.peprotech.com). The cells were challenged by addition of 75–250 ng/ml streptavidin-FasL chimeric protein for 18–24 hours, followed by flow cytometric analysis of apoptosis and death.

Colony-Forming Unit Assay In Vitro
Cells (3 x 104) were plated in 1.2% methylcellulose containing 20% fetal bovine serum, 1% bovine serum albumin, 0.1 mM 2β-ME, 10 U/ml recombinant human erythropoietin (EPO), 20 ng/ml recombinant mouse (rm) SCF, 10 ng/ml rmIL-3, and 10 ng/ml rmGM-CSF (Peprotech) in Iscove's modified Dulbecco's medium (Biological Industries). Colonies exceeding 50 cells (colony-forming unit [CFU]-C) were counted after 7–10 days. All assays were performed in triplicate and were related to simultaneous assays of control cultures containing only the growth factors, to present percentage changes in clonogenic activity. In each experiment, streptavidin-FasL chimeric protein was added at incremental concentrations in the range of 200–1,500 ng/ml or was adsorbed on the surface of the cells via biotinylation. Recombinant human soluble FasL, without cross-linking enhancer (SuperFasL; Alexis Biochemical, San Diego, http://www.axxora.com), was supplemented at a concentration of 5 ng/ml [10, 40]. Caspases 3 and 8 were inhibited by the addition of Z-DEVD-fmk and Z-IETD-fmk (R&D Systems Inc., Abingdon U.K., http://www.rndsystems.com), respectively.

Adsorption of FasL Protein on the Surface of Cells
Nucleated bone marrow cells (BMC) and splenocytes harvested under aseptic conditions were suspended in 5 µM freshly prepared EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL, http://www.piercenet.com) in PBS for 30 minutes at room temperature, as previously described [41, 46, 47]. After being washed twice with PBS, the cells were incubated with streptavidin-FasL chimeric protein (100 ng protein per 1 x 106 cells in PBS), and the efficiency of adsorption was evaluated by flow cytometry using anti-streptavidin and anti-FasL antibodies.

Statistical Analysis
Data are presented as means ± SDs for each experimental protocol. Results in each experimental group were evaluated for reproducibility by linear regression of duplicate measurements. Differences between the experimental protocols were estimated with a post hoc Scheffe t test, and significance was considered at p < .05.


   RESULTS
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Fas and FasL Expression Is Upregulated After Transplantation
Two subsets with distinct hematopoietic reconstituting potentials were isolated from BMC by counterflow centrifugal elutriation [42–45]. Small cells collected at an elutriation flow rate of 25 ml/minute were processed by lineage depletion to yield a fraction (Fr25 lin–) enriched in LTR cells. Large progenitors were collected at the end of the density fractionation procedure in the rotor-off position. Transplantation of these subsets into irradiated (850 rad) syngeneic recipients (CD45.2->CD45.1) results in distinct hematopoietic reconstitution patterns. The majority of mice transplanted with Fr25 lin– cells (8 of 11) succumbed within the period usually observed in myeloablated mice that did not receive cellular transplants. Mice transplanted with R/O cells survived for periods of several weeks, and most of them (5 of 8) failed to establish durable donor-type chimerism. In variance, mice transplanted with both cell populations (LTR + STR) showed durable engraftment and competent hematopoiesis in serial transplants, consistent with prior reports [43].

The expression of the Fas receptor was infrequent in freshly isolated Fr25 lin– and R/O subsets (Fig. 1A). In variance from low levels of FasL expression in R/O cells, 27% ± 4% of the elutriated Fr25 lin– cells were positive for FasL. The expression of Fas receptor and ligand was also assessed in the bone marrow-homed donor cells at 48 hours after transplantation. The bone marrow-homed Fr25 lin– and R/O cells showed marked upregulation of both Fas (Fig. 1B) and FasL, with statistically significant higher levels (p < .001) in R/O progenitors (Fig. 1A). Most cells jointly upregulated the expression of both Fas and FasL (Fig. 1C), suggesting that the cells were insensitive to autocrine apoptosis. In marked variance, Fas and FasL expression was not upregulated in the residual host BMC after irradiation and transplantation (Fig. 1B).


Figure 1
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  		Figure 1. Fas and FasL expression in BM-homed donor cells. (A): Small Fr25 lin– cells and large R/O progenitors isolated by counterflow centrifugal elutriation were evaluated for expression of Fas and FasL (elutriated). The data represent the means of five independent experiments. These subsets were transplanted into irradiated syngeneic hosts (CD45.2->CD45.1; n = 7) and were harvested after 2 days for analysis of Fas and FasL expression (BM-homed). (B): Fas and FasL were acutely upregulated in donor cells (shaded areas) but not in residual host cells (solid line). (C): In most cells, Fas and FasL were jointly upregulated. Data represent means ± SD. Abbreviations: BM, bone marrow; FasL, Fas ligand; lin–, lineage-negative; PE, phycoerythrin; PerCP, Peridinin chlorophyll protein; R/O, rotor-off.

 
Bone Marrow-Homed Donor Cells Are Resistant to Fas-Mediated Apoptosis
The sensitivity of freshly elutriated cells to apoptosis and death was determined in vitro. Cells were incubated in supporting medium and with a FasL chimeric protein that delivers potent apoptotic signals to Fas+ cells [25, 46]. An 18-hour incubation period resulted in upregulation of Fas expression to 18% ± 5% and 42% ± 6% of the Fr25 lin– and R/O cells, respectively. Under these conditions, 10% ± 2% of the Fr25 lin– cells and 22% ± 4% of the R/O cells stained positive for annexin-V (Fig. 2A, medium). Addition of 250 ng/ml FasL protein resulted in apoptosis of 43% ± 6% of the elutriated R/O cells (Fig. 2A, FasL) and a small increase (6% ± 1%) in apoptosis of the Fr25 lin– subset (not shown).


Figure 2
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  		Figure 2. Fas-mediated signaling in hematopoietic progenitors. (A): Elutriated and day 2 BM-homed R/O progenitors were incubated in supporting medium for 18 hours in vitro (medium) and were challenged for apoptosis with 250 ng/ml FasL protein. Apoptosis was assayed by incorporation of annexin-V in flow cytometry (n = 5). (B): Apoptotic death (annexin+) and Fas expression were measured by gating on the donor cells at 2 days after transplantation. All Fr25 lin– Fas+ cells and the majority (75%) of R/O Fas+ cells were resistant to FasL-induced apoptosis. Data represent three independent experiments. (C): Elutriated R/O progenitors were cultured in semisolid methylcellulose medium in the presence of stem cell factor, interleukin 3, granulocyte-macrophage colony-stimulating factor, erythropoietin (naïve), and FasL protein, with and without inhibition of caspase 3 activity with DEVD. Clonogenicity was stimulated at a FasL protein concentration of 500 ng/ml and was completely abolished at a concentration of 1.5 µg/ml. The data summarize four independent experiments of triplicate cultures, and changes in activity were normalized against the control cultures (naïve). Abbreviations: BM, bone marrow; DEVD, Z-DEVD-fmk; FasL, Fas ligand; FITC, fluorescein isothiocyanate; PerCP, Peridinin chlorophyll protein; R/O, rotor-off.

 
Upregulation of the Fas receptor after transplantation suggested that the donor cells might became sensitive to apoptosis. To test their sensitivity, Fr25 lin– and R/O cells transplanted into irradiated syngeneic recipients (CD45.2->CD45.1) were harvested after 48 hours and submitted to the apoptotic challenge in vitro. The bone marrow-homed donor Fr25 lin– cells were largely resistant to the apoptotic challenge, as determined by annexin-V incorporation (not shown). FasL increased the apoptosis of bone marrow-homed R/O cells from 10% ± 2% to 23% ± 4% (p < .005). However, the rates of apoptosis of BM-homed R/O cells were significantly lower than those of freshly elutriated cells (p < .001). These data corroborated our findings in phenotypically characterized HSPC that homed to the host bone marrow after transplantation [25]. Analysis of apoptotic death and Fas expression revealed that virtually all Fr25 lin– Fas+ cells were insensitive to FasL-induced apoptosis, as were the majority (75%) of the R/O Fas+ cells (Fig. 2B).

Fas Ligation Enhances the Clonogenic Activity of Short-Term Reconstituting Progenitors
Insensitivity of the majority of progenitors to Fas-mediated apoptosis called into question whether this receptor has additional functions. To determine whether Fas is involved in trophic signaling, the clonogenic activity of R/O cells stimulated by GM-CSF and EPO was measured in the presence of FasL protein. The clonogenic activity of R/O progenitors increased by 26% ± 6% (p < .005) upon addition of a supralethal concentration of 500 ng/ml FasL protein (Fig. 2C). A FasL protein concentration of 1.5 µg/g completely abolished the clonogenic activity of these progenitors. To evaluate the equilibrium between trophic and apoptotic signals mediated by the Fas receptor, the common effector caspase 3 was inhibited with Z-DEVD-fmk. Caspase 3 inhibition further enhanced the clonogenic activity of progenitors by 75% ± 5% compared with the activity of naïve cells (p < .001). Taken together, these data indicated that Fas mediated dual apoptotic and trophic signals in the subset of early hematopoietic reconstituting progenitors.

The Fas Receptor Mediates Apoptotic and Nonapoptotic Signals
The relative insensitivity of subsets enriched for stem cells and progenitors to FasL-induced apoptosis corroborates similar observations in lin–Sca-1+c-kit+ murine hematopoietic progenitors [25]. Under physiological conditions, cells with hematopoietic reconstituting potential comprise a small number of cells (0.2%–0.5%) embedded in a bulk mass of progenitors and mature cells. To better evaluate the equilibrium between Fas-mediated apoptotic and trophic signaling, we further compared the responses of wBMC and lin– BMC to Fas ligation. The activity of wBMC was progressively suppressed as the concentration of FasL was increased (Fig. 3A). In marked contrast, and in accordance with the effect of FasL on R/O progenitors, the clonogenic activity of lin– BMC was gradually stimulated, reaching a peak increase of ~45% at FasL protein concentrations of 400–600 ng/ml. This behavior was observed both when soluble FasL oligomers were added to the culture medium and when the protein was adsorbed to the cell surface via biotinylation. The protein became toxic to lin– BMC at a threshold concentration of ~1 µg/ml. To determine the effect of FasL on cell viability, suspensions of whole and lin– BMC were incubated for 18 hours. A protein concentration of 200 ng/ml induced apoptosis in 38% ± 6% of naive wBMC (p < .01 vs. 18% ± 4% death in medium), whereas it did not significantly affect the viability of lin– BMC (Fig. 3B).

To ascertain that FasL affected the cells through Fas binding, the clonogenic assays were performed with cells harvested from Fas-defective (lpr) mice. The lpr cells were insensitive to the tropic and apoptotic effects of the FasL protein (Fig. 4A), indicating that modulation of progenitor activity was specifically mediated by Fas receptor ligation. Next, we questioned whether receptor trimerization was required for clonogenic activation. Soluble FasL at a concentration of 5–10 ng/ml [29, 48] did not significantly modify the death and clonogenicity of whole and lin– BMC. Taken together, these data suggested that Fas receptor trimerization was essential for transduction of the growth signals.


Figure 3
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  		Figure 3. Trimerization of the Fas receptor enhances the clonogenic activity of hematopoietic cells. (A): Incubation of whole and lin– BMC with FasL oligomers in semisolid methylcellulose cultures in the presence of stem cell factor, interleukin 3, granulocyte-macrophage colony-stimulating factor, and erythropoietin resulted in a dose-dependent increment in the clonogenic activity of lin– BMC. At high protein concentrations (>1 µg/ml), an abrupt decay in clonogenic activity resembled that observed in whole BMC. The data summarize eight independent experiments. (B): Whole and lin– BMC (5 x 106 cells per milliliter) were incubated for 18 hours in culture medium with and without 200 ng/ml FasL protein. Cell death and apoptosis were determined by incorporation of 7-aminoactinomycin D and annexin-V, respectively, and lineage markers were determined with a cocktail of antibodies. The data summarize five independent experiments. Abbreviations: BMC, bone marrow cells; FasL, Fas ligand; lin–, lineage-negative; wBMC, whole bone marrow cells.

 


Figure 4
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  		Figure 4. Trophic signaling is mediated by the Fas receptor. (A): Whole and lin– BMC from wild-type and Fas-defective (lpr) mice were cultured in semisolid cultures in the presence of stem cell factor, interleukin 3, granulocyte-macrophage colony-stimulating factor, and erythropoietin. In variance from FasL-dependent modulation of the clonogenic activity of wild-type lin– BMC, the lpr cells were unaffected by FasL. The data represent five independent experiments of triplicate cultures. (B): Radiation-conditioned (CD45.1) recipients (850 rad) were transplanted with a mixture of 5 x 105 lin– BMC from wild-type (CD45.2+GFP+) and lpr (CD45.2+GFP–) mice. The levels of donor chimerism were determined at 6 and 20 weeks post-transplantation (n = 12). Abbreviations: BMC, bone marrow cells; GFP, green fluorescent protein; lin–, lineage-negative; wBMC, whole bone marrow cells.

 
To evaluate the physiological role of the Fas receptor in hematopoietic cell transplantation, a competitive engraftment experiment was performed by transplantation of 5 x 105 lin– BMC from both wild-type green fluorescent protein (GFP) mice and lpr (CD45.2) donors into the myeloablated syngeneic (CD45.1) recipients (n = 16). Under these conditions, the chimeras presented 59% ± 5% wild-type (GFP+) (donor) chimerism at 6 weeks, which progressed to 75% ± 6% at 20 weeks post-transplantation (Fig. 4B). Notably, since GFP is not expressed in all bone marrow cells [49], the levels of lpr chimerism may be overestimated. These data indicated that the Fas/FasL interaction is of physiological significance in the process of hematopoietic cell engraftment. Furthermore, these data corroborated the observation that expression of Fas by donor cells did not render them susceptible to apoptosis. On the contrary, Fas might serve for trophic signaling.

Involvement of Caspases in Fas Signaling
We next evaluated possible differential activation of caspases in apoptotic and trophic signaling through the Fas receptor. Inhibition of caspase 3 with Z-DEVD-fmk (Fig. 5A) and of caspase 8 with Z-IETD-fmk (not shown) did not significantly affect the clonogenic activity of lin– BMC and did not interfere with the stimulatory effect of 500 ng/ml FasL. Similarly, inhibition of both caspases had little effect on the clonogenic activity of wBMC in the absence of FasL (Fig. 5B), suggesting that caspase activation was not required for Fas-mediated tropism. However, the inhibition of both caspases restored the decreased clonogenicity of wBMC caused by the presence of FasL protein, with inhibition of caspase 3 having a slightly greater effect than inhibition of caspase 8 (Fig. 5B). The likely explanation for these data was that caspase inhibition reduced Fas-mediated cell death of progenitors. Possibly, the presence of apoptotic/dead cells in the culture affected the overall clonogenic activity. Two experiments were performed to test this mechanism. First, caspases 3 (not shown) and 8 (Fig. 5C) were inhibited in the presence of a toxic dose of FasL protein (1.5 µg/g). In both whole and lin– BMC populations, caspase inhibition caused a partial recovery of the clonogenic activities, showing again that caspase activity was related solely to the apoptotic function of the Fas receptor. Second, an equal number of apoptotic cells were added to cultures of lin– BMC (n = 6). The presence of apoptotic cells reduced the number of colonies by 27% ± 7% (p < .001 vs. control), indicating that viability of the cultured cells affects colony formation. Taken together, these data demonstrated a regulatory function of Fas on the clonogenic activity of progenitors by dual transduction of apoptotic and trophic signals.


Figure 5
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  		Figure 5. The dual function of the Fas receptor in cell growth and death. (A): Inhibition of caspase 3 with DEVD did not significantly affect the clonogenic activity of lin– BMC incubated with and without 500 ng/ml FasL protein. The data summarize four assays performed in triplicate. (B): Inhibition of caspase 3 with DEVD and of caspase 8 with IETD restored the clonogenic activity of whole BMC incubated with 500 ng/ml FasL protein (n = 4). The inhibitors had little effect on clonogenicity. (C): Caspase 3 inhibition with DEVD partially restored the clonogenic activity of whole and lin– BMC incubated with 1.5 µg/g FasL protein (n = 5) by reducing apoptotic death. Abbreviations: BMC, bone marrow cells; DEVD, Z-DEVD-fmk; FasL, Fas ligand; IETD, Z-IETD-fmk; lin–, lineage-negative; wBMC, whole bone marrow cells.

 
Physiological Significance of Fas Expression
Upon homing to the bone marrow of irradiated recipients, the donor cells displayed a remarkable upregulation in expression of Fas and FasL [25, 41]. Following syngeneic transplantation of lin– BMC, 42% ± 5% and 58% ± 7% of the bone marrow-homed cells expressed Fas and FasL, respectively (Fig. 6A). This upregulation was not observed in the residual host cells that survived irradiation. In a previous study, we showed that the expression of ectopic FasL protein on the surface of cells improved engraftment in syngeneic transplants through a Fas-mediated interaction with the marrow stroma [41]. The dissociation of Fas expression from donor cell sensitivity to apoptosis [25] and the superior engraftment of wild-type cells over lpr cells (Fig. 4B) suggest a role of donor cell Fas in the process of engraftment, which is attributed to enhanced clonogenic activity of the progenitors. To directly evaluate whether tropism was of significance in vivo, lin– BMC were exposed to FasL protein for 18 hours prior to transplantation. This incubation period was found to induce Fas expression and induce apoptosis in a fraction of the mature BMC (Fig. 3B). In our hands, the limiting number of lin– cells required to rescue myeloablated syngeneic hosts (950 rad) was 2 x 105 cells; therefore, the mice were infused with 1.5 x 105 BMC. The requirement for a relatively large number of cells is associated with x-ray irradiation, which is more toxic than the standard {gamma}-radiation. In syngeneic transplants (CD45.2->CD45.1) the survival of recipients of FasL-pretreated cells was superior (14 of 20) to that of lin– BMC incubated in medium (9 of 20) (Fig. 6B). The mean survival times were not significantly different: 9.4 ± 2.3 and 11 ± 3.2 days in recipients of naïve and FasL-pretreated cells, respectively. We next evaluated whether the activation of the hematopoietic progenitors by FasL caused extinction of the stem cells with long-term reconstituting potential. Bone marrow cells of the surviving chimeras were harvested 14 weeks after the first transplants and were infused into secondary myeloablated syngeneic recipients (CD45.1). Transplantation of half of the cellular contents of a femur into each one of the secondary recipients resulted in full donor chimerism after 14 weeks, indicating that stem cell self-renewal was preserved after incubation with the proapoptotic ligand. Tertiary transplants performed in the same way showed similar results.


Figure 6
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  		Figure 6. Fas signaling improves the efficiency of early engraftment. (A): Expression of Fas and FasL in donor cells after syngeneic transplantation (CD45.1->CD45.2) of lin– bone marrow cells (BMC) into mice conditioned with 850 rad of total-body irradiation (n = 12). The Fas receptor and its ligand were evaluated by gating on donor cells. (B): Myeloablated hosts (950 rad) were transplanted with 1.5 x 105 BMC after incubation for 18 hours in medium and in the presence of 250 ng/ml FasL protein (n = 20). Irradiated mice that received no cellular transplant served as controls for conditioning (irradiation; n = 10). There was no additional mortality at times longer than the 4 weeks presented here. Abbreviation: FasL, Fas ligand; PE, phycoerythrin; PerCP, Peridinin chlorophyll protein.

 
Under more stringent conditions, we performed allogeneic transplants using cells pre-exposed to FasL protein. Transplantation of equal numbers of viable cells after exposure to FasL protein may be enriched in the absolute number of HSPC, as the apoptotic cells were predominantly within the lin+ subset (Fig. 3B). Approximately 40% of the cells underwent apoptosis during incubation with FasL, compared with ~20% of cells suspended in medium. Therefore, 2 x 105 BMC were incubated with and without FasL protein for 18 hours and were transplanted into irradiated allogeneic hosts (H2Kb->H2Kd). Mice transplanted with cells pre-exposed to FasL showed increased survival rates (10 of 20) compared with recipients of cells incubated in medium (6 of 20). Notably, the protein was not adsorbed on the donor cells; therefore, the superior survival was not mediated by inhibition of alloimmune responses [41]. These data support the concept that exposure to FasL protein improved the radioprotective qualities of the grafted cells.


   DISCUSSION
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
In a developmental system such as the hematopoietic compartment, the stem cells and primitive progenitors should be well protected to guarantee the maintenance of the most important component of the developmental system. Since these cells are often required to operate under hostile conditions, an intuitive assumption would suggest that the progenitors are insensitive to injury signals. We used a transplant model as an extreme situation where hematopoietic cells have to survive and operate within a skewed environment soon after severe damage is inflicted by conditioning. A series of transplant-related events, including conditioning-induced injury to the bone marrow stroma, in situ hematopoietic cell death, and early inflammation, alters the cytokine milieu in the marrow. The environmental cytokine changes include potent stimuli of Fas receptor expression in hematopoietic cells, such as TNF-{alpha} and interferon-{gamma} [10–18]. Under these conditions, donor cells that homed successfully to the host bone marrow displayed a remarkable upregulation of Fas and FasL (Figs. 1, 6A). However, the important parameter revealed by the transplant model system is the insensitivity of stem and progenitor cells to Fas-mediated apoptosis. Inefficient apoptotic signal transduction through Fas indeed preserves the viability of progenitors under these stress conditions. Furthermore, insensitivity to apoptosis endows the hematopoietic progenitors with several significant advantages. First, these cells can use FasL counterattack residual elements of the host immune system and reduce allorejection [41, 50, 51]. Second, the apoptosis-insensitive progenitors have an engraftment advantage over more committed and mature cells that are sensitive to Fas-mediated apoptosis [25]. Third, transduction of trophic signals by Fas in the apoptosis-insensitive murine HSPC enhances the pace of hematopoietic reconstitution in myeloablated recipients. This latter mechanism has previously been underestimated. Our data suggest that in the fine-tuned hematopoietic system, the same receptor that mediates apoptosis in differentiated cells stimulates the activity of progenitors to promote the recovery from aplasia.

The trophic function of Fas in hematopoietic progenitors in vitro was a combined result of two parallel processes: apoptosis and enhanced recruitment of colony-forming cells. The delicate equilibrium between Fas-mediated death and function in the heterogeneous hematopoietic cell compartment was underlined by caspase inhibition. Although inhibition of caspases 3 and 8 reduced FasL-induced apoptotic death of some cells, there was no direct attenuation of clonogenicity. Furthermore, blockade of the proteolytic cascade did not prevent the increase in number of colony-forming cells under FasL stimulation. Taken together, these data imply that a subset of apoptosis-sensitive cells was eliminated by FasL, whereas a subset of Fas-stimulated progenitors was recruited to perform clonogenic activity. Otherwise, it would be difficult to dissociate between these processes, because addition of dead cells to the culture decreased the clonogenic activities. Thus, evaluation of the impact of death receptors in clonogenic assays also has to account for indirect factors, such as the presence of apoptotic cells, in addition to the direct consequences of receptor ligation. Therefore, exposure of the cultures to FasL did not directly cause a true reduction in number of colony-forming cells; rather, it recruited more progenitors to perform clonogenic activity, underscoring the insensitivity of these cells to Fas-mediated apoptosis. This attributes Fas an important regulatory role in this developmental system, where a receptor that predominantly signals growth in the proximal stages converts into a major negative regulator in distal development. In the distal stages of differentiation of immunohematopoietic cells, death receptors of the TNF superfamily in general, and Fas in particular, are powerful negative regulators that control the size of the expanding clones [30–37].

The data presented here revoke a detrimental role of Fas in hematopoietic progenitor engraftment in vivo. The robust upregulation of Fas in bone marrow-homed donor cells [25, 41] obviated the need for pretransplant culture to induce the expression of Fas and further corroborates the observation that the hematopoietic reconstituting cells resided exclusively in the Fas-positive fraction of murine [37] and human [52] progenitors. Culture of progenitors and manipulation with TNF and various FasL agonists prior to transplantation is particularly problematic, as it frequently results in reduced engraftability in parallel to Fas upregulation. Such cultures alter the engraftment of hematopoietic progenitors in a variety of ways, some of which are independent of Fas [53, 54]. For example, murine cells exposed to cytokines are often deficient in homing to the host bone marrow, decreasing the efficiency of hematopoietic reconstitution [55].

The most convincing supporting evidence for a causal relationship was the reversal of defective engraftment by blocking anti-Fas antibodies and soluble FasL [21, 22, 37, 38], suggesting that apoptotic signaling converges with the differential engraftment of cells positioned in various phases of the cell cycle. Here, we attempted to dissociate the behaviors of cycling progenitors and putative LTR stem cells positioned in G0/G1 using counterflow elutriation [56]. Mitotically quiescent and slowly cycling cells positioned in the G0/G1 cycle phase engraft with superior efficiency compared with cells positioned in other phases [57, 58]. Under culture conditions, the progenitors are induced to progress into active phases of the cell cycle, resulting in reduced engraftment [57, 58]. We recently demonstrated that Fas expression is induced both by active cycling and differentiation after donor cell homing to the host bone marrow [25]. It is possible that the engraftment of some apoptosis-sensitive (fast-cycling) progenitors with limited reconstituting potential was impaired; however, the engraftment of radioprotective cells was rather enhanced. We interpreted this behavior as a competitive advantage of the repopulating primitive progenitors over more differentiated cells [25]. Similar results have been reported for TNF-{alpha}, shown to equally suppress fast-cycling progenitors of wild-type and lpr mice in a Fas-independent manner [37]. In the Fr25 lin– fraction enriched in candidate LTR stem cells, hematopoietic reconstitution persisted in sequential transplants following exposure to FasL and after decoration with ectopic FasL protein (M. Pearl-Yafe, N. Askenasy, unpublished data). Unlike the apparent inhibition of stem cell self-renewal under stimulated culture conditions [37], our findings demonstrate that the reconstituting capacity of Fas+ LTR cells was, at least, not impaired by the expression of Fas.

The current data demonstrate that activation and not inhibition of the Fas receptor was responsible for progenitor stimulation, as trimerization of the Fas receptor was a requisite for both tropic and apoptotic signaling. It is important to dissociate between the inhibitory (antiapoptotic) effects of soluble FasL and the stimulatory (apoptotic and tropic) functions of membranous FasL [59]. Under selected conditions, soluble FasL serves as an apoptotic factor that prevents Fas receptor activation through trimerization [59, 60]. Soluble FasL did not significantly attenuate the number of murine colony-forming cells, consistent with prior work showing no significant effect of soluble FasL and activating Fas antibodies (Jo2) on the clonogenicity of murine and human progenitors [22, 29, 37, 52]. Only oligomerization of the Fas receptor resulted in transduction of the signals. Similarly, Fas trimerization was required to achieve costimulatory responses in T cells activated with anti-CD3, indicating that oligomerization of the receptor is essential to enhance T-cell proliferation [61].

Several series of studies has addressed growth-related functions of Fas and the intracellular signal transduction pathways associated with the death receptors (reviewed in [19, 62, 63]). Nonapoptotic signaling through Fas and TNF receptors is important in a variety of cells, including lymphocytes, regenerating hepatocytes, osteoclasts, and mesenchymal stem cells [61, 64–67]. In T lymphocytes, the Fas and TNF-{alpha} receptors have long been recognized as important components of functional and mitotic activation [61, 64, 65, 68]. The dual stimulatory and restrictive effects of TNF-{alpha} [39 are easier to explain, considering the differential consequences of TNF-R1 and TNF-R2 activation [19, 63]. However, there is evidence that TNF-R2 itself transduces dual apoptotic and growth signals in T cells [68]. Likewise, we show that the Fas receptor transduces trophic and apoptotic signals in hematopoietic progenitors. A candidate proximal point of divergence between growth and death signals is the formation of the death-inducing complex. Although resting hepatocytes are very sensitive to apoptosis induced via the Fas and TNF-R1 receptors, their activation supports the process of regeneration [69, 70]. Deficient liver regeneration in lpr mice was not apparent in lpr.cg mice [70], which possess a competent Fas receptor and bear a mutation that prevents activation of the Fas-associated death domains [71]. Thus, differential engagement of the Fas-associated death domains may dissociate between trophic and apoptotic signals. This appears to be a relatively late and adaptive process in hematopoietic cells, because Fas-associated death domains and caspase 8 deficits lead to death in the embryonic phase [72].

Caspase inhibition did not directly attenuate the clonogenic potential of the progenitors, placing the Fas-related tropic signal transduction pathway proximal to caspase 8. These data corroborate other reports of nonapoptotic signaling triggered by Fas ligation through pathways that do not involve the caspase proteolytic cascade [73–76]. A point of divergence between tropic and apoptotic signals may be FLIP, which is elevated in hematopoietic progenitors and endows them with a survival advantage [29, 77]. FLIP controls a checkpoint between caspase 8 activation and stimulation of the antiapoptotic nuclear factor {kappa}B pathway [62]. However, it is unlikely that this is the sole regulator of sensitivity to apoptosis, as caspases are involved in a series of nonapoptotic activities that affect development and function of differentiated immunohematopoietic cells. For example, Fas-mediated signaling participates in physiological T lymphocyte stimulation, and caspase activation is a physiological early event in lymphocyte proliferation [61, 64, 65, 72]. Accordingly, inhibition of the effector caspases abrogates the stimulatory effects of Fas in T cells [61]. The deletion of caspase 8 was also shown to arrest myelomonocytic progenitor function and prevent macrophage differentiation and death [78]. Additional experiments will be required to characterize the differential signaling pathways of Fas-mediated tropism and apoptosis in hematopoietic progenitors.

Experimental Considerations
Our experimental approach differs in several respects from previous studies. The most significant factor was the dissociation between the robust upregulation of Fas and FasL in bone marrow-homed donor cells and their sensitivity to apoptosis [25, 41]. These findings triggered the examination of the role of Fas in progenitor cell function and engraftment without enforcing its expression by ex vivo stimulation in the presence of TNF-{alpha} [11–18, 22, 37, 38, 52]. Possibly, some of the allegations of a detrimental effect of Fas on HSPC engraftment originated from a quantitative relationship between its expression and Fas-independent events triggered by the culture conditions. Second, in our attempt to dissociate between the proapoptotic and nonapoptotic consequences of Fas signaling in hematopoietic reconstituting cells, the target populations were the lin– BMC and elutriated subsets with distinct functional potentials. Further purification and phenotypic identification of the cells would be confusing because isolation of pure stem cell populations is practically unachievable. Despite the robust upregulation of Fas in the Fr25 lin– subset enriched in long-term reconstituting cells, their in vitro assessment was not possible because these cells retain functional quiescence for extended periods of time. In addition, selection of hematopoietic progenitors according to their Fas expression is a biased approach because this is a dynamic process that occurs in bone marrow cells under a variety of physiological and in vitro conditions (M. Pearl-Yafe, N. Askenasy, unpublished observations).

Third, a chimeric protein composed of streptavidin and noncleavable FasL was used to challenge for apoptosis. The use of soluble isoforms of FasL and activating anti-Fas antibodies for induction of apoptosis [17, 22, 37, 38, 52, 60] imposes interpretation difficulties because soluble FasL has both pro- and antiapoptotic functions under selected conditions [59]. Although concentrations in the range of 10–50 ng/ml of commercially available FasL were shown to induce effective apoptosis in A20 cells, we found similar activities at concentrations of 25–100 ng/ml of the chimeric protein [41, 46, 47]. A concentration exceeding 250 ng/ml caused death of ~40% of the mature bone marrow cells [25]. Since this is a fusion protein that lacks the metalloproteinase cleavage site, it is difficult to quantitatively relate the efficiencies in apoptotic and tropic effects. In particular, it is difficult to assess whether the concentrations of FasL shown here to exert tropic effects are within the physiological concentration in the bone marrow microenvironment. The most significant differences in clonogenic activities were reported using a Jo2 anti-Fas antibody, which varies from the FasL protein used here also in its toxicity to the liver [46, 50, 70]. Finally, we used two cytokines for cells in suspension (SCF and TPO) and four cytokines for clonogenic assays in semisolid medium (SCF, IL-3, GM-CSF, and EPO), whereas numerous other combinations have previously been used, including TNF-{alpha}. Various cytokine combinations influence the expression of the Fas receptor and maturation of the apoptotic cascade in stem and progenitor cells in different ways, influencing the sensitivity of cells to apoptosis [79].

In summary, the Fas receptor transduces stimulatory signals in hematopoietic progenitors that are of physiological significance in the transplant setting. The process of progenitor recruitment to clonogenic activity is mediated by trimerization of the Fas receptor without activation of effector caspases. This receptor evolves as a powerful and efficient regulator of hematopoietic progenitors that controls an important junction of function and death. The results are of physiological significance to a number of pathologies associated with increased apoptosis of normal and malignant hematopoietic cells. Peripheral cytopenias in patients suffering from myelodysplastic syndromes are often accompanied by marrow hyperplasia, suggesting increased intra- and extramedullary apoptosis of hematopoietic cells [80]. Fas expression in CD34+ cells correlated neither with the severity of cytopenia nor with the sensitivity to apoptosis [80], suggesting that upregulated expression may be part of the effort to recover from the cytopenic state. Furthermore, the resistance of primitive hematopoietic progenitors to Fas-mediated apoptosis may have significant implications in view of the suppressive role of this receptor in leukemogenic transformation in myeloid progenitor cells [24]. It remains to be determined whether the tropic functions of Fas also apply to malignant blasts.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The chimeric FasL protein technology used in this study is licensed from the University of Louisville by ApoImmune, Inc. (Louisville, KY), for which H.S. serves as Chief Scientific Officer; H.S. and E.S.Y. have significant equity in the company. H.S. and E.S.Y. own stock in, have acted as consultants to, have performed contract work for, have served as officers or members of the Board for, and have a financial interest in ApoImmune, Inc.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
The guidance and help of Drs. Saul J. Sharkis and Michael Collector in establishment of the centrifugal elutriation procedure is gratefully acknowledged. We thank Natalia Binkovsky and Ela Zuzovsky for outstanding technical support. This work was funded by Grant 2003276 from the United States-Israel Binational Science Foundation (to N.A., I.Y., H.S., and E.S.Y.), a grant from the Frankel Trust for Experimental Bone Marrow Transplantation (to J.S. and I.Y.), ADA Grant 1-05-JF-56, NIH Grant R21 HL080108-01 (to E.S.Y.) NIH Grant R01-AI47864, NIH Grant R21 AI057903 (to H.S.), and Juvenile Diabetes Research Fund innovative Grant 5-2005-1102 (to N.A.).


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 Introduction
 Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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